An innovative intramedullary bone graft harvesting concept as a fundamental component of scaffold-guided bone regeneration: A preclinical in vivo validation

Background The deployment of bone grafts (BGs) is critical to the success of scaffold-guided bone regeneration (SGBR) of large bone defects. It is thus critical to provide harvesting devices that maximize osteogenic capacity of the autograft while also minimizing graft damage during collection. As an alternative to the Reamer-Irrigator-Aspirator 2 (RIA 2) system – the gold standard for large-volume graft harvesting used in orthopaedic clinics today – a novel intramedullary BG harvesting concept has been preclinically introduced and referred to as the ARA (aspirator + reaming-aspiration) concept. The ARA concept uses aspiration of the intramedullary content, followed by medullary reaming-aspiration of the endosteal bone. This concept allows greater customization of BG harvesting conditions vis-à-vis the RIA 2 system. Following its successful in vitro validation, we hypothesized that an ARA concept-collected BG would have comparable in vivo osteogenic capacity compared to the RIA 2 system-collected BG. Methods We used 3D-printed, medical-grade polycaprolactone-hydroxyapatite (mPCL-HA, wt 96 %:4 %) scaffolds with a Voronoi design, loaded with or without different sheep-harvested BGs and tested them in an ectopic bone formation rat model for up to 8 weeks. Results Active bone regeneration was observed throughout the scaffold-BG constructs, particularly on the surface of the bone chips with endochondral bone formation, and highly vascularized tissue formed within the fully interconnected pore architecture. There were no differences between the BGs derived from the RIA 2 system and the ARA concept in new bone volume formation and in compression tests (Young's modulus, p = 0.74; yield strength, p = 0.50). These results highlight that the osteogenic capacities of the mPCL-HA Voronoi scaffold loaded with BGs from the ARA concept and the RIA 2 system are equivalent. Conclusion In conclusion, the ARA concept offers a promising alternative to the RIA 2 system for harvesting BGs to be clinically integrated into SGBR strategies. The translational potential of this article Our results show that biodegradable composite scaffolds loaded with BGs from the novel intramedullary harvesting concept and the RIA 2 system have equivalent osteogenic capacity. Thus, the innovative, highly intuitive intramedullary harvesting concept offers a promising alternative to the RIA 2 system for harvesting bone grafts, which are an important component for the routine translation of SGBR concepts into clinical practice.

-3 -Supplementary Table 2. Detailed explanations and descriptions on the morphological assessment of mineralized samples, including the evaluation of active bone regeneration.Scanning electron microscopy provides very high spatial resolution and a wide field of view, allowing the characterization of bone microstructure.In vivo performance of biomedical implant materials can be assessed in detail using SEM [1,2] and particularly the evaluation of the osteocyte network within implanted biomaterials [3,4,5].Moreover, osteocyte LCN can be well depicted on resin-cast etched ground sections [5,6] through osteocyte etching (removal of the inorganic phase with phosphoric acid (H3PO4) and digestion of the organic phase with sodium hypochlorite (NaOCl)).

Focus
( Modified GT staining offers the distinct benefit of overall tissue morphology due to sharply distinguishing mature bone matrix, immature new bone matrix, and calcified cartilage [7].Standard laboratory protocols [8,9] were applied.The rhodamine molecule is small enough (~0.9 nm) to penetrate the cortical bone via the canaliculi [10] and binds to osseous surfaces.Sample preparation, staining procedure, and imaging techniques have been described in detail elsewhere [11].Resin blocks were used to visualize osteocytes and the LCN with rhodamine staining through CLSM.Briefly, the samples were incubated in 70% ethanol for 5 days, 80% An innovative intramedullary bone graft harvesting concept as a fundamental component of scaffold-guided bone regeneration: A preclinical in vivo validation Laubach et al.
-4 - ethanol for 5 days, and 90% ethanol for 9 days.Next, rhodamine 6G (#R4127-5G, Sigma) was diluted in 100% ethanol (250 mL 100% ethanol mixed with 1.25 g of rhodamine 6G for 30 min) and the samples were incubated protected from light for 3 days.The samples were then cleared in 2 changes of xylene for 4 h each and incubated in resin pre-infiltration solution for 7 days, followed by incubation in infiltration solution for 7 days (for details of solution, see [8]).The samples were resin embedded under vacuum and left to polymerize at 4 °C for 7 days.Briefly, the rhodamine-stained and polymethyl methacrylate (PMMA) resin-embedded samples were imaged with fluorescence CLSM (Leica TCS SP8 DLS Multiphoton, Wetzlar, Germany), equipped with an oil immersion lens (Leica, HCX PL APO 40× NA 1.25 oil) using argon laser light (λexcitation = 514 nm / λemission = 550-650 nm).The confocal microscope was equipped with a Mai Tai HP multiphoton laser, which was used to visualize the SHG signal of fibrillar collagen of the same resin blocks.Second-harmonic generation excitation wavelength was set at 910 nm and detection wavelength at 450-460 nm.A strong SHG signal is evident in regions with dense and well-aligned collagen fibrils perpendicular to the incoming light.
An innovative intramedullary bone graft harvesting concept as a fundamental component of scaffoldguided bone regeneration: A preclinical in vivo validation Laubach et al.
-5 -Supplementary Table 3. Overview of the number of samples of the respective experimental group that were used for the different analysis methods.μCT, micro-computed tomography.

Analysis
) Confocal fluorescence imaging of xylenol orange to qualitatively confirm bone regeneration Resin blocks of fluorochrome-labelled tissue were protected from intense light during processing, stored in the dark, and 50 µm thick ground sections were used for further analysis.The 50 μm ground resin sections stained with xylenol orange were imaged at the maximum intensity projections of the z-stacks.Images were acquired at 2.5 µm intervals along the z-axis on a Nikon TiE inverted widefield microscope (Nikon, Tokyo, Japan) through a Nikon Plan Apo 10x, 0.45 NA DIC L objective.The fluorescence filters used were a Nikon FITC filter set (excitation 465-495 nm, 505 nm dichroic beam splitter, emission 515-555 nm) and a Nikon TRITC filter set (excitation 528-552 nm, 565 nm dichroic beam splitter, emission 558-632 nm).Exposure times were 2 ms for the FITC channel and 30 ms for the FITC channel.

Table 1 .
An innovative intramedullary bone graft harvesting concept as a fundamental component of scaffold-guided bone regeneration: A preclinical in vivo validation Laubach et al.Primary antibodies and protocol specifications for immunohistochemistry markers used in this study.
An innovative intramedullary bone graft harvesting concept as a fundamental component of scaffold-guided bone regeneration: A preclinical in vivo validation Laubach et al.